Antibody formulations and methods

ABSTRACT

Antibody formulations and methods useful for prophylaxis or treatment of amyloidosis, including AA amyloidosis and AL amyloidosis.

RELATED APPLICATIONS

Priority is claimed to U.S. Provisional Application No. 61/551,406,filed 25 Oct. 2011, which is incorporated by reference herein in itsentirety.

TECHNICAL FIELD

The invention resides in the technical fields of immunology andmedicine.

BACKGROUND OF THE INVENTION

Amyloidosis is a general term that describes a number of diseasescharacterized by the existence of pathological forms of amyloidproteins, often involving extracellular deposition of protein fibrils,which form numerous “amyloid deposits” or “amyloid plaques,” which mayoccur in local sites or systematically. These deposits or plaques arecomposed primarily of a naturally occurring soluble protein or peptide,assembled into extensive insoluble deposits 10-100 μm in diameter in avariety of tissue sites. The deposits are composed of generally lateralaggregates of fibrils that are approximately 10-15 nm in diameter.Amyloid fibrils produce a characteristic apple green birefringence inpolarized light, when stained with Congo Red dye. Generally, thefibrillar composition of these deposits is an identifying characteristicfor the various forms of amyloid disease.

The peptides or proteins forming the plaque deposits are often producedfrom a larger precursor protein. More specifically, the pathogenesis ofamyloid aggregates such as fibril deposits generally involvesproteolytic cleavage of an “abnormal” precursor protein into fragmentsthat aggregate into anti-parallel β pleated sheets.

Systemic amyloidoses are a complex group of diseases caused by tissuedeposition of misfolded proteins that result in progressive organdamage. The most common type, AL amyloidosis or primary amyloidosis,involves a hematological disorder caused by clonal plasma cells thatproduce misfolded immunoglobulin light chains. Overproduction ofmisfolded light chain by plasma cells results in deposits of abnormal ALprotein (amyloid), in the tissues and organs of individuals with ALamyloidosis. Clinical features of AL amyloidosis include a constellationof symptoms and organ dysfunction that can include cardiac, renal, andhepatic dysfunction, gastrointestinal involvement, neuropathies andmacroglossia. The mechanisms by which amyloidogenic immunoglobulin lightchains result in organ dysfunction are not well characterized, however,it is hypothesized that both amyloid deposits and prefibrillaraggregates may contribute to cytotoxic effects on organs observed inpatients with AL amyloidosis. AL amyloidosis is a disease entity of itsown, although AL amyloidosis can occur concurrently in a small subset(up to 15%) of patients with multiple myeloma.

AL amyloidosis is a rare disorder with an estimated incidence of 8 in1,000,000 people. Only 1200 to 3200 new cases of AL amyloidosis arereported each year in the United States. Two thirds of patients with ALamyloidosis are male and less than 5% of patients are under 40 years ofage. Both the causes and origins of AL amyloidosis remain poorlyunderstood.

Current treatment of patients with AL amyloidosis is aimed at reducingor eliminating the bone marrow disorder, i.e. the plasma cells that areresponsible for producing the light chains, thereby limiting or haltingthe production of amyloid. The most aggressive treatment options includestem cell transplant and high-dose chemotherapy for those patients whocan tolerate it. Other treatment regimens include combinations of drugsoften used to treat hematological malignancies, such as melphalan,prednisone, dexamethasone and proteosome inhibitors such as bortezomib,in an attempt to reduce light chain production. There are no currentlyapproved treatments for AL amyloidosis that directly target potentiallytoxic forms of the amyloidogenic proteins.

A different form of systemic amyloidosis, AA amyloidosis or secondaryamyloidosis, occurs “secondarily” as a result of other illness, such aschronic inflammatory diseases (for example, rheumatoid arthritis andankylosing spondylitis) or chronic infections (for example, tuberculosisor osteomyelitis). In secondary amyloidosis, the depositing amyloidprotein is amyloid A protein, derived from an acute-phase protein serumamyloid A. The treatment of secondary amyloidosis is directed attreating the underlying illness.

Thus, there is a need for therapies to treat AA amyloidosis and ALamyloidosis, which directly target the pathological amyloid fibrils. Thepresent invention provides pharmaceutical formulations of 2A4 and 7D8antibodies, and chimeric and humanized versions thereof, which show highaffinity binding to both AL and AA amyloids due to a shared immunogenicepitope of the pathological forms of these proteins.

SUMMARY OF THE INVENTION

The present invention provides antibody formulations useful forprophylaxis and treatment of amyloid disease. In one aspect of theinvention, a pharmaceutical formulation comprises (a) a chimeric orhumanized version of antibody 2A4 (ATCC Accession Number PTA-9662) or ofantibody 7D8 (ATCC Accession Number PTA-9468), or fragment thereof,which specifically competes for binding to antigen with 2A4 or 7D8,and/or which is directed to an epitope comprising AEDS (SEQ ID NO: 18),wherein the antibody is present at a concentration within the range fromabout 1 mg/mL to about 100 mg/mL; (b) histidine buffer present at aconcentration within the range from about 20 mM to about 30 mM; (c)trehalose present at a concentration within the range from about 210 mMto about 250 mM; and (d) polysorbate 20 present at a concentrationwithin the range from about 0.005% to about 0.05% by weight; wherein theformulation is characterized by a pH within the range from about 6 toabout 7. For example, representative formulations of the inventioncomprise an antibody having a light chain variable region comprising anamino acid sequence set forth as SEQ ID NO: 4 and/or a heavy chainvariable region comprising an amino acid sequence set forth as SEQ IDNO: 5. More particularly, such a formulation can comprise an antibodyhaving a light chain comprising an amino acid sequence set forth as SEQID NO: 13 and a heavy chain comprising an amino acid sequence set forthas any one of SEQ ID NO: 14-16, for example, an antibody having a lightchain comprising an amino acid sequence set forth as SEQ ID NO: 13 and aheavy chain comprising an amino acid sequence set forth as SEQ ID NO:15.

Additional representative formulations of the invention comprise (a) anantibody having a light chain variable region comprising threecomplementarity determining regions set forth as SEQ ID NOs: 6, 7, and8, and a heavy chain variable region comprising three complementarityregions set forth as SEQ ID NOs: 9, 10, and 11; and (b) an antibodyhaving a light chain variable region comprising three complementaritydetermining regions set forth as SEQ ID NOs: 12, 7, and 8, and a heavychain variable region comprising three complementarity regions set forthas SEQ ID NOs: 9, 10, and 11.

In representative formulations of the invention, the antibody is presentat a concentration within the range from about 5 mg/mL to about 15 mg/mL(e.g., about 10 mg/mL), or present at a concentration within the rangefrom about 25-75 mg/mL (e.g., 50 mg/mL).

In other representative formulations of the invention, histidine bufferis present at a concentration of about 25 mM. The histidine buffer cancomprise L-histidine and L-histidine HCl monohydrate. For example,L-histidine can be used at a concentration within the range from about16 mM to about 22 mM and L-histidine HCl monohydrate can be used at aconcentration within the range from about 4 mM to about 8 mM.

In other representative formulations of the invention, trehalose ispresent at a concentration of about 230 mM.

Prepared as described herein, representative formulations of theinvention (a) are characterized by an osmolality of about 300 mOsm/kg;(b) comprise less than about 10% of the antibody present as an aggregatein the formulation; (c) further comprise a bulking agent; (d) aresterile; and/or (e) are stable upon freezing and thawing.

In one aspect of the invention, a formulation comprises (a) an antibodycomprising a light chain comprising an amino acid sequence set forth asSEQ ID NO: 13 and a heavy chain comprising an amino acid sequence setforth as any one of SEQ ID NOs: 14-16, and which is present at aconcentration of about 10 mg/mL; (b) a histidine buffer present at aconcentration of about 25 mM; (c) trehalose present at a concentrationof about 230 mM; (d) polysorbate 20 present at a concentration of about0.2 g/L; and (e) a pH of about 6.5.

In another aspect of the invention, a pharmaceutical formulationcomprises (a) an antibody, which is antibody 2A4 (ATCC Accession NumberPTA-9662), antibody 7D8 (ATCC Accession Number PTA-9468), or a chimericor humanized version of antibody 2A4 or of antibody 7D8, or fragmentthereof, which specifically competes for binding to antigen with 2A4 or7D8, and/or which is directed to an epitope comprising AEDS (SEQ ID NO:18), wherein the antibody is present at a concentration within the rangefrom about 50 mg/mL to about 100 mg/mL; (b) a buffer; (c) a non-reducingsugar; and (d) a non-ionic surfactant. In particular examples, theantibody of the disclosed formulations comprises a light chaincomprising an amino acid sequence set forth as SEQ ID NO: 13 and a heavychain comprising an amino acid sequence set forth as SEQ ID NOs: 15.

In another aspect of the invention, the antibody formulations arelyophilized. For example, a representative lyophilized formulation cancomprise: (a) a humanized version of antibody 2A4 (ATCC Accession NumberPTA-9662) or antibody 7D8 (ATCC Accession Number PTA-9468) or antigenbinding fragment thereof; (b) histidine; (c) trehalose; and (d)polysorbate 20. Lyophilized formulations can have a pH of between about6 to about 7 when reconstituted, such as pH 6.5 when reconstituted.Lyophilized formulations typically comprise about 100 mg to about 1000mg of the antibody. Lyophilized formulations typically comprisepolysorbate 20 at a concentration within the range from about 0.005% toabout 0.05% by weight. Following reconstitution, the lyophilizedformulations yield an aqueous solution, for example, an aqueous solutioncomprising: (a) an antibody comprising a light chain comprising an aminoacid sequence set forth as SEQ ID NO: 13 and a heavy chain comprising anamino acid sequence set forth as any one of SEQ ID NOs: 14-16, and whichis present at a concentration of about 10 mg/mL; (b) a histidine bufferpresent at a concentration of about 25 mM; (c) trehalose present at aconcentration of about 230 mM; (d) polysorbate 20 present at aconcentration of about 0.2 g/L; and (e) a pH of about 6.5. Arepresentative lyophilized formulation comprises about 100 mg of theantibody following reconstitution with sterile water.

Also provided are nucleic acids encoding antibodies used to prepare thedisclosed formulations. For example, such nucleic acids include nucleicacids comprising nucleotide sequences encoding an antibody light chainof SEQ ID NO: 13 and nucleic acids comprising nucleotide sequencesencoding an antibody heavy chain of any one of SEQ ID NOs: 14-16. Forexample, the nucleotide sequences set forth as SEQ ID NO: 19 and SEQ IDNO: 20 (which is identical to SEQ ID NO: 19 and further includes asequence encoding a signal peptide) each encode the humanized 2A4 lightchain of SEQ ID NO: 13. As another example, the nucleotide sequences setforth as SEQ ID NO: 22 and SEQ ID NO: 23 (which is identical to SEQ IDNO: 22 and further includes a sequence encoding a signal peptide) eachencode the humanized 2A4 heavy chain of SEQ ID NO: 15.

For the production of antibodies, the disclosed nucleic acids may beincluded in a vector, either singly or in combination (e.g., acombination of a nucleic acid encoding a humanized 2A4 light chain and anucleic acid encoding a humanized 2A4 heavy chain). For example, avector can comprise a nucleic acid comprising a nucleotide sequenceencoding any one of SEQ ID NOs: 13-16, 21, and 24; a nucleic acidcomprising the nucleotide sequence of any one of SEQ ID NOs: 19-20 and22-23, or combinations thereof. Representative vectors of the inventioninclude (a) a vector comprising a nucleic acid sequence encoding ahumanized 2A4 light chain set forth as SEQ ID NO: 13 or 21 and ahumanized 2A heavy chain set forth as SEQ ID NO: 15 or 24; (b) a vectorcomprising a nucleic acid having the nucleotide sequence of SEQ ID NO:19 and a nucleic acid having the nucleotide sequence of SEQ ID NO: 22;and (c) a vector comprising a nucleic acid having the nucleotidesequence of SEQ ID NO: 20 and a nucleic acid having the nucleotidesequence of SEQ ID NO: 23.

Also provided are host cells (e.g., CHO cells) having stablyincorporated into their genomes one or more of the nucleic acidsdisclosed herein. For example, a host cell can comprise in its genome astably integrated nucleic acid comprising a nucleotide sequence encodingany one of SEQ ID NOs: 13-16, 21, and 24; a stably integrated nucleicacid comprising the nucleotide sequence of any one of SEQ ID NOs: 19-20and 22-23, or combinations thereof. Representative host cells of theinvention include (a) host cells comprising a nucleic acid sequenceencoding a humanized 2A4 light chain set forth as SEQ ID NO: 13 or 21and a humanized 2A heavy chain set forth as SEQ ID NO: 15 or 24; (b)host cells comprising a nucleic acid having the nucleotide sequence ofSEQ ID NO: 19 and a nucleic acid having the nucleotide sequence of SEQID NO: 22; and (c) host cells comprising a nucleic acid having thenucleotide sequence of SEQ ID NO: 20 and a nucleic acid having thenucleotide sequence of SEQ ID NO: 23.

The present invention also provides methods of preparing pharmaceuticalformulations. In one aspect of the invention, such a method comprises(a) culturing mammalian cells having stably incorporated into theirgenome nucleic acids encoding the light and heavy chains of a murine,chimeric or humanized 2A4 antibody or of a murine, chimeric or humanized7D8 antibody so that the cells secrete the antibody into the cellculture media, and purifying the antibody from the cell culture media;(b) and preparing a formulation comprising (i) a chimeric or humanizedversion of antibody 2A4 (ATCC Accession Number PTA-9662) or of antibody7D8 (ATCC Accession Number PTA-9468), or fragment thereof, thatspecifically competes for binding to antigen with 2A4 or 7D8, whereinthe antibody is present at a concentration within the range from about 1mg/mL to about 100 mg/mL; (ii) histidine buffer present at aconcentration within the range from about 20 mM to about 30 mM; (iii)trehalose present at a concentration within the range from about 210 mMto about 250 mM; and (iv) polysorbate 20 present at a concentrationwithin the range from about 0.005% to about 0.05% by weight; wherein theformulation is characterized by a pH within the range from about 6 toabout 7. For example, in one aspect of the invention, mammalian cellshaving stably incorporated into their genomes nucleic acids encoding thelight and heavy chains of a humanized 2A4 antibody are cultured.Mammalian cells useful for this purpose include (a) host cells havingstably incorporated into their genomes a nucleic acid sequence encodinga humanized 2A4 light chain set forth as SEQ ID NO: 13 or 21 and ahumanized 2A heavy chain set forth as SEQ ID NO: 15 or 24; (b) hostcells having stably incorporated into their genomes a nucleic acidhaving the nucleotide sequence of SEQ ID NO: 19 and a nucleic acidhaving the nucleotide sequence of SEQ ID NO: 22; and (c) host cellshaving stably incorporated into their genomes a nucleic acid having thenucleotide sequence of SEQ ID NO: 20 and a nucleic acid having thenucleotide sequence of SEQ ID NO: 23. In some aspects of the invention,the disclosed methods of preparing a pharmaceutical formulation includethe additional step of evaluating at least one property of antibody inthe formulation, such as physical stability, chemical stability, and/orbiological activity.

Still further provided are methods of therapeutically orprophylactically treating a human patient having or at risk of havingamyloidosis characterized by the presence of amyloid protein fibrils,the method comprising administering to the patient an effective dosageof a formulation of the invention. Patients amenable to treatment havean amyloid disease such as amyloid A amyloidosis, which is characterizedby the presence of amyloid A protein fibrils, or AL amyloidosis, whichis characterized by the presence of amyloid light chain-type proteinfibrils. Patients having AL amyloidosis may also suffer from anassociated dyscrasia of the B lymphocyte lineage, for example amalignancy such as multiple myeloma.

The disclosed therapeutic and prophylactic treatment methods includecombination therapies (i.e., administration of the disclosed antibodyformulations with one or more additional drug substances) to therebyelicit synergistic results. The two or more drug substances areadministered simultaneously or sequentially in any order, i.e., aformulation of the invention is administered prior to administration ofa second drug substance, concurrently with a second drug substance, orsubsequent to administration of a second drug substance. For example, aformulation of the invention can be administered concurrently orconsecutively in combination with melphalan. As another example, aformulation of the invention can be administered concurrently orconsecutively in combination with one or more of bortezomib, melphalan,lenalidomide and carfilzomib.

In accordance with the disclosed therapeutic and prophylactic treatmentmethods, formulations of the invention can be administered in multipledosages, for example, at a frequency in a range of about daily to aboutannually, such as at a frequency in a range of about every other week toabout every three months, or such as once a month. In one aspect, anantibody formulation of the invention is administered intravenously at adose in a range from about 10 mg to about 5000 mg drug substance. Forexample, a formulation can be administered at a dose in a range fromabout 30 mg to about 2500 mg humanized 2A4 drug substance at a frequencyin a range of about every other week to about every other month.Representative dosages used in the disclosed methods include 30 mg, 100mg, 300 mg, 1000 mg, 2000 mg, and 2500 mg of humanized 2A4 drugsubstance.

In one aspect of the invention, a method of therapeutically orprophylactically treating a human patient having or at risk for havinglight chain (AL) amyloidosis characterized by the presence of amyloidfibrils, deposits or prefibrillar aggregates, comprises administering tothe patient an effective dosage of a pharmaceutical formulationcomprising: (a) an antibody comprising a light chain comprising an aminoacid sequence set forth as SEQ ID NO: 13 and a heavy chain comprising anamino acid sequence set forth as any one of SEQ ID NOs: 14-16, and whichis present at a concentration of about 10 mg/mL; (b) a histidine bufferpresent at a concentration of about 25 mM; (c) trehalose present at aconcentration of about 230 mM; (d) polysorbate 20 present at aconcentration of about 0.2 g/L; and (e) a pH of about 6.5. In such amethod, the dosage is typically from about 0.5 mg/kg to about 30 mg/kgof the antibody (e.g., about 0.5 mg/kg to about 8 mg/kg, or about 8mg/kg to about 30 mg/kg) administered intravenously or subcutaneously ata frequency of from about weekly to about quarterly (e.g., once every 28days).

The present invention further provides a pharmaceutical productcomprising: (a) a vial comprising about 100 mg antibody in powder form;(b) instructions for reconstitution of the antibody; and (c)instructions for preparing the reconstituted antibody for infusion,wherein (i) the antibody comprises a light chain comprising an aminoacid sequence set forth as SEQ ID NO: 13 and a heavy chain comprising anamino acid sequence set forth as any one of SEQ ID NOs: 14-16; and (ii)the reconstitution instructions require reconstitution with water forinjection to an extractable volume of 10 mL.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B show various humanized 2A4 antibody light chain and heavychain sequences. Bold and underlining, consensus sequence for N-linkedglycosylation.

FIG. 2 shows murine 2A4 and 7D8 light chain variable region (VL) andheavy chain variable region (VH) sequences. Double underlining, leadersequence; underlining, complementarity determining region (CDR)sequences.

FIG. 3 shows humanized 2A4 version 3 light chain variable region (VL)and heavy chain variable region (VH) sequences. Lower case, backmutations.

FIGS. 4A-4B show nucleic acid sequences encoding humanized 2A4 version 3heavy chain (FIG. 4A) and heavy chain (FIG. 4B) sequences. Singleunderline, leader sequence; no underline, variable region; doubleunderline, constant region.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides antibody formulations useful forprophylaxis and treatment of amyloid disease. In one aspect of theinvention, a pharmaceutical formulation comprises (a) a chimeric orhumanized version of antibody 2A4 (ATCC Accession Number PTA-9662) or ofantibody 7D8 (ATCC Accession Number PTA-9468), or fragment thereof,which specifically competes for binding to antigen with 2A4 or 7D8,and/or which is directed to an epitope comprising AEDS (SEQ ID NO: 18),wherein the antibody is present at a concentration within the range fromabout 1 mg/mL to about 100 mg/mL; (b) histidine buffer present at aconcentration within the range from about 20 mM to about 30 mM; (c)trehalose present at a concentration within the range from about 210 mMto about 250 mM; and (d) polysorbate 20 present at a concentrationwithin the range from about 0.005% to about 0.05% by weight; wherein theformulation is characterized by a pH within the range from about 6 toabout 7.

In one aspect of the invention described herein, humanized 2A4 is anIgG1, kappa isotype version of murine 2A4. In the course of specificitycharacterization of humanized 2A4, the antibody was found to also reactwith high affinity and in a conformation-dependent manner with lightchain in light chain amyloid fibrils, but not with free light chain incirculation.

The present invention provides methods for intravenous infusion ofhumanized 2A4 and/or humanized 7D8 antibodies to target misfoldedamyloid protein in patients with AA amyloidosis and AL amyloidosis. Somehumanized 2A4 antibodies specifically bind to pathologic amyloid formsof AL and SAA but do not bind to the parent molecules from which thesepathologic forms are derived (SAA, native immunoglobulin light chain[LC], intact immunoglobulin [Ig]).

I. Pharmaceutical Formulations and Products

I.A. Characteristics

Provided herein are pharmaceutical formulations comprising a chimeric orhumanized version of antibody 2A4 (ATCC Accession Number PTA-9662) or ofantibody 7D8 (ATCC Accession Number PTA-9468), or fragment thereof, thatspecifically competes for binding to antigen (i.e., human AA or ALprotein) with 2A4 or 7D8, respectively, and/or that is directed to theepitope AEDS (SEQ ID NO: 18). Also provided are pharmaceuticalformulations comprising murine antibody 2A4 or murine antibody 7D8, orfragments thereof. The antibody is present at a concentration within therange from about 1 mg/mL to about 100 mg/mL. The formulation ischaracterized by a pH within the range from about 6 to about 7 andcomprises a histidine buffer at a concentration within the range fromabout 20 mM to about 30 mM, trehalose at a concentration within therange from about 210 mM to about 250 mM; and polysorbate 20 at aconcentration within the range from about 0.005% to about 0.05% byweight.

The term “humanized immunoglobulin” or “humanized antibody” refers to animmunoglobulin or antibody that includes at least one humanizedimmunoglobulin or antibody chain (i.e., at least one humanized light orheavy chain). The term “humanized immunoglobulin chain” or “humanizedantibody chain” (i.e., a “humanized immunoglobulin light chain” or“humanized immunoglobulin heavy chain”) refers to an immunoglobulin orantibody chain (i.e., a light or heavy chain, respectively) having avariable region that includes a variable framework region substantiallyfrom a human immunoglobulin or antibody and complementarity determiningregions (CDRs) (e.g., at least one CDR, preferably two CDRs, morepreferably three CDRs) substantially from a non-human immunoglobulin orantibody, and further includes constant regions (e.g., at least oneconstant region or portion thereof, in the case of a light chain, andpreferably three constant regions in the case of a heavy chain). Theterm “humanized variable region” (e.g., “humanized light chain variableregion” or “humanized heavy chain variable region”) refers to a variableregion that includes a variable framework region substantially from ahuman immunoglobulin or antibody and complementarity determining regions(CDRs) substantially from a non-human immunoglobulin or antibody.

The phrase “substantially from a human immunoglobulin or antibody” or“substantially human” means that, when aligned to a human immunoglobulinor antibody amino sequence for comparison purposes, the region shares atleast 80-90%, preferably 90-95%, more preferably 95-99% identity (i.e.,local sequence identity) with the human framework or constant regionsequence, allowing, for example, for conservative substitutions,consensus sequence substitutions, germline substitutions, backmutations,and the like. The introduction of conservative substitutions, consensussequence substitutions, germline substitutions, backmutations, and thelike, is often referred to as “optimization” of a humanized antibody orchain. The phrase “substantially from a non-human immunoglobulin orantibody” or “substantially non-human” means having an immunoglobulin orantibody sequence at least 80-95%, preferably 90-95%, more preferably,96%, 97%, 98%, or 99% identical to that of a non-human organism, e.g., anon-human mammal.

Accordingly, all regions or residues of a humanized immunoglobulin orantibody, or of a humanized immunoglobulin or antibody chain, exceptpossibly the CDRs, are substantially identical to the correspondingregions or residues of one or more native human immunoglobulinsequences. The term “corresponding region” or “corresponding residue”refers to a region or residue on a second amino acid or nucleotidesequence which occupies the same (i.e., equivalent) position as a regionor residue on a first amino acid or nucleotide sequence, when the firstand second sequences are optimally aligned for comparison purposes.

In some formulations, the antibody comprises a light chain variableregion comprising an amino acid sequence set forth as any one of SEQ IDNOs: 1, 2, or 4. In some formulations, the antibody comprises a heavychain variable region comprising an amino acid sequence set forth as SEQID NO: 3 or 5. In some formulations, the antibody comprises a lightchain variable region comprising an amino acid sequence set forth as anyone of SEQ ID NOs: 1, 2, or 4 and a heavy chain variable regioncomprising an amino acid sequence set forth as SEQ ID NO: 3 or 5. Insome formulations, the antibody comprises a light chain variable regioncomprising an amino acid sequence set forth as SEQ ID NO: 1 and a heavychain variable region comprising an amino acid sequence set forth as SEQID NO: 3. In some formulations, the antibody comprises a light chainvariable region comprising an amino acid sequence set forth as SEQ IDNO: 4 and a heavy chain variable region comprising an amino acidsequence set forth as SEQ ID NO: 5. In some formulations, the antibodycomprises a light chain variable region comprising an amino acidsequence set forth as SEQ ID NO: 2 and a heavy chain variable regioncomprising an amino acid sequence set forth as SEQ ID NO: 3.

In some formulations, the antibody comprises a light chain variableregion comprising three complementarity determining regions set forth asSEQ ID NOs: 6, 7, and 8, and a heavy chain variable region comprisingthree complementarity regions set forth as SEQ ID NOs: 9, 10, and 11. Inother formulations, the antibody comprises a light chain variable regioncomprising three complementarity determining regions set forth as SEQ IDNOs: 12, 7, and 8, and a heavy chain variable region comprising threecomplementarity regions set forth as SEQ ID NOs: 9, 10, and 11.

In other formulations of the present invention, the antibody compriseslight chain and heavy chain variable regions of a murine, chimeric, orhumanized 2A4 antibody, or of a murine, chimeric, or humanized 7D8antibody, as described in U.S. Pat. No. 7,928,203 and PCT InternationalPublication No. WO 2009/086539, each of which is incorporated herein byreference in its entirety, and the light chain and heavy chain variableregion sequences described in the referenced patent and publication arespecifically incorporated by reference herein.

In some formulations, the antibody comprises a light chain comprising anamino acid sequence set forth as SEQ ID NO: 13 or 21 and a heavy chaincomprising an amino acid sequence set forth as any one of SEQ ID NOs:14-16 and 24. The antibody can include, or not include, the leadersequences of the above-noted light chain and heavy chain amino acidsequences.

In other formulations, the antibody is a fragment of a 2A4 or 7D8antibody, including chimeric and humanized versions thereof, such as aFab fragment, a Fab′ fragment, a F(ab′)₂ fragment, a Fv fragment or aScFv fragment.

In some aspects of the invention, the antibody specifically binds toaggregated amyloid protein without specifically binding to monomericamyloid protein (e.g., at least a 10-fold and usually at least 100-foldlower specific binding affinity for monomeric forms of the amyloidprotein).

In some formulations, the antibody is present at a concentration withinthe range from about 5 mg/mL to about 100 mg/mL. In some formulations,the antibody is present at a concentration within the range from about 5mg/mL to about 15 mg/mL. In some formulations, the antibody is presentat a concentration within the range from about 25 mg/mL to about 75mg/mL. For example, the antibody may be present at a concentration ofabout 10 mg/mL, or present at a concentration of about 50 mg/mL. Theantibody may be present in a sterile liquid dosage form of about 50mg/vial to about 500 mg/vial, or greater. For example, the antibody maybe present in a sterile liquid dosage form of about 100 mg/vial.

Antibodies used in the disclosed formulations can be coupled with atherapeutic moiety, such as a cytotoxic agent, a radiotherapeutic agent,an immunomodulator, a second antibody (e.g., to form an antibodyheteroconjugate), or any other biologically active agent thatfacilitates or enhances the activity of a chimeric or humanized 2A4 or achimeric or humanized 7D8 antibody. Representative therapeutic moietiesinclude agent known to be useful for treatment, management, oramelioration of amyloid disease or symptoms of amyloid disease.

Antibodies used in the disclosed formulations can also be coupled with adetectable label, for example, as useful for diagnosing an amyloiddisorder, for monitoring progression of amyloid disease, and/or forassessing efficacy of treatment. Antibodies formulated as described areparticularly useful for performing such determinations in subjectshaving or being susceptible to AA amyloidosis or AL amyloidosis, or inappropriate biological samples obtained from such subjects.Representative detectable labels that may be coupled or linked to ahumanized 2A4 antibody or humanized 7D8 antibody include variousenzymes, such as horseradish peroxidase, alkaline phosphatase,beta-galactosidase, or acetylcholinesterase; prosthetic groups, suchstreptavidin/biotin and avidin/biotin; fluorescent materials, such asbut umbelliferone, fluorescein, fluorescein isothiocynate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;luminescent materials, such as luminol; bioluminescent materials, suchas luciferase, luciferin, and aequorin; radioactive materials, such asbut not limited to iodine (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I) carbon (¹⁴C), sulfur(⁵S), tritium (³H), indium (¹¹⁵In, ¹¹³In, ¹¹²In, ¹¹¹In) and technetium(⁹⁹Tc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd),molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd,¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Ru, ⁹⁷Ru,⁶⁸Ge, ⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr, ³²P, ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn, ⁷⁵Se, ¹¹³Sn, and¹¹⁷Tin; positron emitting metals using various positron emissiontomographies, nonradioactive paramagnetic metal ions, and molecules thatare radiolabelled or conjugated to specific radioisotopes.

Therapeutic moieties and/or detectable substances may be coupled orconjugated directly to a murine, chimeric or humanized 2A4 antibody or amurine, chimeric or humanized 7D8 antibody, or indirectly, through anintermediate (e.g., a linker) using techniques known in the art. Seee.g., Arnon et al., “Monoclonal Antibodies For Immunotargeting Of DrugsIn Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy,Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstromet al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2ndEd.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987);Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: AReview”, in Monoclonal Antibodies 84: Biological And ClinicalApplications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis,Results, And Future Prospective Of The Therapeutic Use Of RadiolabeledAntibody In Cancer Therapy”, in Monoclonal Antibodies For CancerDetection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press1985), and Thorpe et al., Immunol. Rev., 1982, 62:119-58.

Antibodies used in the disclosed formulations also include modifiedforms of murine, chimeric or humanized 2A4 antibodies, or murine,chimeric or humanized 7D8 antibodies, which have increased in vivohalf-lives relative to the corresponding unmodified antibodies. Suchmodified forms may be prepared, for example, by glycosylation,acetylation, pegylation, phosphorylation, amidation, derivatization byknown protecting/blocking groups, proteolytic cleavage, linkage to acellular ligand or other protein, etc. As one example, representativemethods for antibody half-life extension are described in PCTInternational Publication No. WO 02/060919.

The present invention encompasses antibody formulations having stabilityat 38° C.-42° C. as assessed by high performance size exclusionchromatography (HPSEC) for at least about 30 days, formulations havingstability at 20° C.-24° C. for at least about 1 year, and formulationshaving stability at 2° C.-4° C. for at least about 3 years. Moreparticularly, the disclosed formulations exhibit low to undetectablelevels of antibody aggregation and/or fragmentation, or a low orundetectable increase of antibody fragmentation and/or aggregation abovean initial level (e.g., less than about 10% aggregation. A formulationhaving low to undetectable levels of fragmentation contains at leastabout 80%, 85%, 90%, 95%, 98%, or 99%, of the total protein, forexample, in a single peak as determined by high performance sizeexclusion chromatography (HPSEC), or in two (2) peaks (one correspondingto each of the antibody heavy chains and antibody light chains) byreduced Capillary Gel Electrophoresis (rCGE), representing thenon-degraded antibody, and containing no other single peaks having morethan 5%, more than 4%, more than 3%, more than 2%, more than 1%, or morethan 0.5% of the total protein each. A formulation having low toundetectable levels of aggregation contains no more than about 15%, nomore than about 10%, no more that about 5%, no more than about 4%, nomore than about 3%, no more than about 2%, no more than about 1%, or nomore than about 0.5% aggregation by weight protein as measured by highperformance size exclusion chromatography (HPSEC). For example, in someformulations, less than about 10% of the anti-amyloid antibody ispresent as an aggregate. Stable formulations of the invention also showlittle or no loss of biological activity(ies) of a chimeric or humanized2A4 or chimeric or humanized 7D8, for example binding affinitymeasurable by ELISAs and/or additional functional assays, such as atleast about at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 98%, or 99% of an initial measurable value of a given activity.

The histidine buffer may be present in some formulations at aconcentration of about 25 mM. In some formulations, the histidine buffercomprises L-histidine and L-histidine HCl monohydrate. For example, insome formulations, L-histidine is present at a concentration within therange from about 16 mM to about 22 mM and L-histidine HCl monohydrate ispresent at a concentration within the range from about 4 mM to about 8mM.

In some formulations, trehalose is present at a concentration from about210 mM to about 250 mM, for example, about 230 mM. In some formulations,a different non-reducing sugar is used, such as sucrose, mannitol, orsorbitol.

In some formulations, polysorbate 20 is present at a concentrationwithin the range of about from about 0.005% to about 0.05% by weight,for example, 0.005%, 0.01%, 0.015%, 0.02%, 0.025%, 0.03%, 0.035%, 0.04%,0.045%, or 0.05%. Alternatively, in some formulations, polysorbate 20 ispresent at a concentration within the range of about from about 0.05g/L, 0.1 g/L, 0.15 g/L, 0.2 g/L, 0.25 g/L, 0.3 g/L, 0.35 g/L, 0.4 g/L,0.45 g/L, or 0.5 g/L. Some formulations include polysorbate 20 at aconcentration of 0.2 g/L.

Some formulations are characterized by a pH within the range of about6-7, for example, a pH of 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8,6.9, or 7.0. Some formulations have a pH of about 6.5.

Some formulations are characterized by an osmolality of about 300mOsm/kg.

A bulking agent may also be included some formulations.

Typically, the formulations are sterile, for example, as accomplished bysterile filtration using a 0.2 μm or a 0.22 μm filter. The formulationsof the invention are also generally stable upon freezing and thawing.

Optionally, formulations of the invention may further comprise otherexcipients, such as saccharides, polyols, and amino acids (e.g.,arginine, lysine, and methionine). In other aspects, the presentinvention also provides formulations substantially free of surfactant,inorganic salts, additional sugars, and/or other excipients, i.e., lessthan about less than 0.0005%, less than 0.0003%, or less than 0.0001% ofsuch compounds.

An exemplary formulation comprises an antibody comprising a light chaincomprising an amino acid sequence set forth as SEQ ID NO: 13 and a heavychain comprising an amino acid sequence set forth as any one of SEQ IDNOs: 14, 15, or 16, which is present at a concentration of about 10mg/mL, a histidine buffer present at a concentration of about 25 mM,trehalose present at a concentration of about 230 mM; polysorbate 20present at a concentration of about 0.2 g/L, and a pH of about 6.5.

I.B. Preparation of Pharmaceutical Formulations

The present invention also provides methods of preparing pharmaceuticalformulations. In one aspect of the invention, such a method comprises(a) culturing mammalian cells having stably incorporated into theirgenome nucleic acids encoding the light and heavy chains of murineantibody 2A4 (ATCC Accession Number PTA-9662) or of antibody 7D8 (ATCCAccession Number PTA-9468), or of chimeric or humanized versionsthereof, so that the cells secrete the antibody into the cell culturemedia, and purifying the antibody from the cell culture media; (b) andpreparing a formulation comprising (i) the purified antibody present ata concentration within the range from about 1 mg/mL to about 100 mg/mL;(ii) histidine buffer present at a concentration within the range fromabout 20 mM to about 30 mM; (iii) trehalose present at a concentrationwithin the range from about 210 mM to about 250 mM; and (iv) polysorbate20 present at a concentration within the range from about 0.005% toabout 0.05% by weight; wherein the formulation is characterized by a pHwithin the range from about 6 to about 7.

In some aspects of the invention, the disclosed methods of preparing apharmaceutical formulation include the additional step of evaluating atleast one property of antibody in the formulation selected from thegroup consisting of the physical stability, chemical stability andbiological activity.

For example, in one aspect of the invention, mammalian cells arecultured for the production of antibodies, wherein the mammalian cellshave stably incorporated into their genomes nucleic acids encoding thelight and heavy chains of a humanized 2A4 antibody. Mammalian cellsuseful for this purpose include (a) host cells having stablyincorporated into their genomes a nucleic acid sequence encoding ahumanized 2A4 light chain set forth as SEQ ID NO: 13 or 21 and ahumanized 2A heavy chain set forth as SEQ ID NO: 15 or 24; (b) hostcells having stably incorporated into their genomes a nucleic acidhaving the nucleotide sequence of SEQ ID NO: 19 and a nucleic acidhaving the nucleotide sequence of SEQ ID NO: 22; and (c) host cellshaving stably incorporated into their genomes a nucleic acid having thenucleotide sequence of SEQ ID NO: 20 and a nucleic acid having thenucleotide sequence of SEQ ID NO: 23.

For the production of antibodies, the disclosed nucleic acids areincluded in a vector. In some examples, the vector contains the nucleicacid encoding murine 2A4 of 7D8 antibody, or a chimeric or humanizedversion thereof, operably linked to a suitable control sequence capableof effecting the expression of the DNA in a host cell. Such controlsequences include a promoter to effect transcription (e.g., aconstitutive promoter or inducible promoter as known in the art), anoptional operator sequence to control such transcription, a sequenceencoding suitable mRNA ribosome binding sites, enhancers,polyadenylation signals, and sequences to control the termination oftranscription and translation. The vector may be a plasmid, a phageparticle (e.g., a viral vector such as adenovirus,adeno-associated-virus, retrovirus, herpes virus, vaccinia virus,lentivirus, poxvirus and cytomegalovirus vectors), or simply a genomicinsert. Once transformed into a suitable host, the antibody nucleicacids may integrate into the genome of the host, or the vector mayreplicate and function independently of the host genome.

The disclosed nucleic acids are included in a vector either singly or incombination (e.g., a combination of a nucleic acid encoding an antibodylight chain and a nucleic acid encoding an antibody heavy chain). Forexample, a vector can comprise a nucleic acid comprising a nucleotidesequence encoding any one of SEQ ID NOs: 13-16, 21, or 24; a nucleicacid comprising the nucleotide sequence of any one of SEQ ID NOs: 19-20and 22-23, or combinations thereof. Representative vectors of theinvention include (a) a vector comprising a nucleic acid sequenceencoding a humanized 2A4 light chain set forth as SEQ ID NO: 13 and ahumanized 2A heavy chain set forth as SEQ ID NO: 15; (b) a vectorcomprising a nucleic acid having the nucleotide sequence of SEQ ID NO:19 and a nucleic acid having the nucleotide sequence of SEQ ID NO: 22;and (c) a vector comprising a nucleic acid having the nucleotidesequence of SEQ ID NO: 20 and a nucleic acid having the nucleotidesequence of SEQ ID NO: 23.

Host cells useful for preparing antibody formulations of the inventioninclude mammalian cells, including cells of human origin, such as monkeykidney cells, human embryonic kidney cells, baby hamster kidney (BHK)cells, Chinese hamster ovary cells (CHO) cells, mouse sertoli cells,human cervical carcinoma (HeLa) cells, canine kidney cells, human lungcells, human liver cells, mouse mammary tumor cells, and NS0 cells. Forexample, a host cell can comprise in its genome a stably integratednucleic acid comprising a nucleotide sequence encoding any one of SEQ IDNOs: 13-16, 21, and 24; a stably integrated nucleic acid comprising thenucleotide sequence of any one of SEQ ID NOs: 19-20 and 22-23, orcombinations thereof. Representative host cells of the invention include(a) host cells comprising a nucleic acid sequence encoding a humanized2A4 light chain set forth as SEQ ID NO: 13 or 21 and a humanized 2Aheavy chain set forth as SEQ ID NO: 15 or 24; (b) host cells comprisinga nucleic acid having the nucleotide sequence of SEQ ID NO: 19 and anucleic acid having the nucleotide sequence of SEQ ID NO: 22; and (c)host cells comprising a nucleic acid having the nucleotide sequence ofSEQ ID NO: 20 and a nucleic acid having the nucleotide sequence of SEQID NO: 23.

In another aspect of the invention, a chimeric or humanized 2A4 antibodyor a chimeric or humanized 7D8 antibody is prepared by chemicalsynthesis and then used in the disclosed formulations.

Antibodies used to prepare the disclosed formulations are typicallyisolated or purified, i.e., substantially free of cellular material orother contaminating proteins from the cells in which they are produced,or substantially free of chemical precursors or other chemicals whenchemically synthesized. For example, an antibody that is substantiallyfree of cellular material includes preparations of the antibody havingless than about 30%, 25%, 20%, 15%, 10%, 8%, 5%, 2%, 1%, 0.5%, 0.1%, orless (by dry weight) of contaminating protein. When an antibody isrecombinantly produced, it is also substantially free of culture mediumsuch that culture medium represents less than about 30%, 25%, 20%, 15%,10%, 8%, 5%, 2%, 1%, 0.5%, 0.1%, or less, of the volume of the proteinpreparation. When an antibody is produced by chemical synthesis, it ispreferably substantially free of or separated from chemical precursorsor other chemicals involved in the synthesis of the protein.Accordingly, such antibody preparations have less than about 30%, 25%,20%, 15%, 10%, 8%, 5%, 2%, 1%, 0.5%, 0.1%, or less (by dry weight) ofchemical precursors or compounds other than the antibody drug substance.Purification of recombinantly expressed antibody can utilize any of anumber of methods known in the art, such as, for example, affinitychromatography, acid treatment, depth filtration, anion exchangechromatography, cation exchange chromatography, nanofiltration,ultrafiltration, dialysis and diafiltration.

The purified antibody drug substance can be adjusted to a solutioncomprising any of the formulations described herein, diluted to thedesired concentration and stored until ready for use. Optionally, theformulation can be stored in concentrated form until ready for use.Liquid formulations can be stored in frozen form, under refrigeration orat room temperature, depending upon their stability profile, which canbe determined empirically. In some instances a further filtration stepis applied. Some of the formulations described herein may be lyophilizedand stored in powder form. Lyophilized formulations can be stored infrozen form, under refrigeration or at room temperature, depending upontheir stability profile, which can be determined empirically. Forexample, the lyophilized formulations can be stored at a temperature ofabout 2° C. to 8° C. In such cases, the formulation would bereconstituted prior to administration to a patient to yield a liquidformulation having the antibody and excipients present in theconcentrations described herein. In some cases, the formulation isreconstituted in sterile water. In some cases, the formulation isreconstituted and added to an infusion bag for administration to thepatient. The reconstituted formulation can be stored under refrigerationor at room temperature prior to administration to a patient for a timeconsistent with the stability profile. Lyophilization and reconstitutiontechniques are known in the art and described in the Examples.

Thus, the present invention also encompasses pharmaceutical productscomprising lyophilized antibody drug substance and instructions forreconstitution and use. For example, a representative pharmaceuticalproduct can comprise: (a) a vial comprising about 100 mg antibody inpowder form; (b) instructions for reconstitution of the antibody; and(c) instructions for preparing the reconstituted antibody for infusion,wherein (i) the antibody comprises a light chain comprising an aminoacid sequence set forth as SEQ ID NO: 13 and a heavy chain comprising anamino acid sequence set forth as any one of SEQ ID NOs: 14-16; and (ii)the reconstitution instructions require reconstitution with water forinjection to an extractable volume of 10 mL.

II. Methods of Diagnosis and Treatment

Also provided are methods of therapeutically or prophylacticallytreating a human patient having or at risk of having amyloidosischaracterized by the presence of amyloid protein fibrils, the methodcomprising administering to the patient an effective dosage of any ofthe formulations described herein.

II.A. Subjects Amenable to Diagnosis and Treatment

Humanized 2A4 drug substance is to be used for the treatment of systemicamyloidosis, such as amyloidoses involving either amyloid light chain ALor amyloid A (AA) proteins. Systemic amyloidoses are a complex group ofdiseases caused by tissue deposition of misfolded proteins that resultin progressive organ damage. The most common type, AL amyloidosis orprimary amyloidosis, involves a hematological disorder caused by clonalplasma cells that produce misfolded immunoglobulin light chains.Overproduction of misfolded light chain by plasma cells results indeposits of abnormal AL protein (amyloid), in the tissues and organs ofindividuals with AL amyloidosis. Clinical features of AL amyloidosisinclude a constellation of symptoms and organ dysfunction that caninclude cardiac, renal, and hepatic dysfunction, GI involvement,neuropathy's and macroglossia. A different form of systemic amyloidosis,AA amyloidosis or secondary amyloidosis, occurs “secondarily” as aresult of other illness, such as chronic inflammatory diseases (forexample, rheumatoid arthritis and ankylosing spondylitis) or chronicinfections (for example, tuberculosis or osteomyelitis). In secondaryamyloidosis, the depositing amyloid protein is amyloid A protein,derived from an acute-phase protein serum amyloid A.

Peripheral amyloidosis is be amenable to this type of amyloid-specificimmunotherapy through antibody targeting of a neo-epitope that has beenidentified in AA amyloid, as well as in AL amyloid. Studies in animalmodels of both AA and AL have demonstrated that significant positivetherapeutic effects may be possible at reasonable doses of antibody.

Subjects or patients amenable to treatment using the disclosed antibodyformulations include individuals at risk of disease but not showingsymptoms, as well as patients presently showing symptoms of amyloiddisease. Therefore, the present methods can be administeredprophylactically to the general population without the need for anyassessment of the risk of the subject patient. For example, the presentmethods are especially useful for individuals who do have a knowngenetic risk autoimmune disorders. Such individuals include those havingrelatives who have experienced this disease and those whose risk isdetermined by analysis of genetic or biochemical markers. As anotherexample, patients suffering from AA amyloidosis can be asymptomatic fora prolonged period of time, such that clinical diagnosis of AAamyloidosis is often delayed or missed until the amyloid deposits areextensive. For those patients who are symptomatic, it is estimated thatonly 53% of the cases are diagnosed. See e.g., L.E.K. Consulting,Independent Market Research (2003). Prophylactic administrationdisclosed antibody formulations may reduce the incidence of AAamyloidosis.

The present methods are especially useful for individuals who do have aknown risk of, are suspected to have, or have been diagnosed with AAamyloidosis or AL amyloidosis. Such individuals include but are notlimited to those having chronic inflammatory diseases, inheritedinflammatory diseases, and chronic microbial infections, such asrheumatoid arthritis, juvenile chronic arthritis, ankylosingspondylitis, psoriasis, psoriatic arthropathy, Reiter's syndrome, AdultStill's disease, Behcet's syndrome, Crohn's disease, FamilialMediterranean Fever, leprosy, tuberculosis, bronchiectasis, decubitusulcers, chronic pyelonephritis, osteomyelitis, Whipple's disease,myeloma, macroglobulinemia, immunocyte dyscrasia, monoclonal gammopathy,occult dyscrasia. Chronic inflammatory and infectious conditions areprerequisite to the development of AA amyloidosis and AL amyloidosismanifested by local nodular amyloidosis can be associated with chronicinflammatory diseases. Individuals who do have known risk of AAamyloidosis also include but are not limited to those having malignantneoplasms as Hodgkin's lymphoma, renal carcinoma, carcinomas of gut,lung and urogenital tract, basal cell carcinoma, and hairy cellleukemia. Additionally, individuals with known risk of AA amyloidosisalso include but are not limited to those having lymphoproliferativedisorders such as Castleman's Disease. Some of such patients have AAamyloidosis characterized by the presence of amyloid A protein fibrils.Some of such patients have AL amyloidosis characterized by the presenceof amyloid light chain-type protein fibrils. Some patients have systemicorgan dysfunction attributed to AL amyloidosis, including dysfunction ofthe heart, kidney, liver, peripheral nervous system, gastrointestinalsystem, autonomic nervous system, lung, and/or soft tissue or lymphaticsystem.

Patients amenable to treatment also include those patients who havereceived, are currently receiving, or will later receive an alternatetherapy, for treatment of amyloid disease or an associated condition,such as, inflammatory diseases, chronic microbial infections, malignantneoplasms, inherited inflammatory diseases, and lymphoproliferativedisorders. For example, patients may also receive or have received oneor more of the therapeutic agents identified herein with respect tocombination therapies. As a particular example, patients suffering fromAL may also receive or have received bortezomib, melphalan, lenalidomideand/or carfilzomib. For those patients who have previously receivedalternate therapies for the treatment of amyloid disease, such therapiesmay or may not have been successful by the relevant clinical measures.

II.B. Treatment Regimes

As used herein, the terms “treat” and “treatment” refer to thealleviation or amelioration of one or more symptoms or effectsassociated with the disease, prevention, inhibition or delay of theonset of one or more symptoms or effects of the disease, lessening ofthe severity or frequency of one or more symptoms or effects of thedisease, and/or increasing or trending toward desired outcomes asdescribed herein.

Desired outcomes of the treatments disclosed herein vary according tothe amyloid disease and patient profile and are readily determinable tothose skilled in the art. Generally, desired outcomes include measurableindices such as reduction or clearance of pathologic amyloid fibrils,decreased or inhibited amyloid aggregation and/or deposition of amyloidfibrils, and increased immune response to pathologic and/or aggregatedamyloid fibrils. Desired outcomes also include amelioration of amyloiddisease-specific symptoms. For example, desired outcomes for thetreatment of AL amyloidosis include a decrease in the incidence orseverity of known symptoms, including organ dysfunction, peripheral andautonomic neuropathy, carpal tunnel syndrome, macroglossia, restrictivecardiomyopathy, arthropathy of large joints, immune dyscrasias,myelomas, as well as occult dyscrasias. As another example, desiredoutcomes for the treatment of AA include a decrease in associatedinflammation, arthritis, psoriasis, microbial infection, malignancy, orsymptoms of other preexisting or coexisting disease to which the AAamyloidosis is secondary.

Desired outcomes of the disclosed therapies are generally quantifiablemeasures as compared to a control or baseline measurement. As usedherein, relative terms such as “improve,” “increase,” or “reduce”indicate values relative to a control, such as a measurement in the sameindividual prior to initiation of treatment described herein, or ameasurement in a control individual or group. A control individual is anindividual afflicted with the same amyloid disease as the individualbeing treated, who is about the same age as the individual being treated(to ensure that the stages of the disease in the treated individual andthe control individual are comparable), but who has not receivedtreatment using the disclosed antibody formulations. In this case,efficacy of the disclosed antibody formulations is assessed by a shiftor trend away from measurable indices in the untreated control.Alternatively, a control individual is a healthy individual, who isabout the same age as the individual being treated. In this case,efficacy of the disclosed antibody formulations is assessed by a shiftor trend toward from measurable indices in the healthy control. Changesor improvements in response to therapy are generally statisticallysignificant and described by a p-value less than or equal to 0.1, lessthan 0.05, less than 0.01, less than 0.005, or less than 0.001 may beregarded as significant.

In both asymptomatic and symptomatic patients, treatment according tothe disclosed methods can begin at any time before or after thediagnosis of the underlying AA or AL amyloid diseases. Treatmenttypically entails multiple dosages over a period of time. Treatment canbe monitored by assaying antibody, or employing radiolabeled SAPScintigraphy over time. If the response falls, a booster dosage may beindicated. The response of patients with AL amyloidosis to treatment canbe monitored by assessing cardiac markers, such as NT-proBNP and/ortroponin, serum creatine, and/or alkaline phosphatase; by performingserum free light chain (SFLC) assays, quantitative immunoglobulinassays, biopsies, serum protein electrophoresis (SPEP), urine proteinelectrophoresis (UPEP), serum, urine immunofixation electrophoresis(IFE), and/or organ imaging techniques. An exemplary complete response(CR) can be determined from response criteria including negative IFE ofserum and urine, normal K/λ ration and/or <5% plasma cells in bonemarrow. An exemplary very good partial response (VGPR) can be determinedfrom a dFLC of <40 mg/L. An exemplary partial response (PR) can bedetermined from a dFLC decrease of ≧50%. In the kidney, a response totreatment can be determined, for example, from a ≧50% reduction(e.g., >0.5 g/24 hours) in 24 hour urine protein excretion in theabsence of either a reduction in eGFR of ≧25% or an increase in serumcreatine of ≧0.5 mg/dL. In the liver, a response to treatment can bedetermined, for example, from a ≧50% reduction in initially elevatedalkaline phosphatase or a ≧2 cm reduction in liver size on CT scan orMRI. In the heart, a response to treatment can be determined, forexample, from a ≧30% and 300 ng/L reduction in NT-proBNP in patientswith baseline of NT-proBNP of >650 ng/L.

The antibody formulation can be administered intravenously in dosageranges from about 10 mg to about 5000 mg for the patient in question,such as, for example, about 10 mg, about 30 mg, about 100 mg, about 300mg, about 1000 mg, about 2000 mg, or about 2500 mg. The antibodyformulation can also be administered intravenously in dosage ranges fromabout 0.1 mg/kg to about 50 mg/kg, or from about 0.5 mg/kg to about 30mg/kg, of the host body weight. For example, dosages can be about 0.5mg/kg body weight, about 1.0 mg/kg, about 1.5 mg/kg, about 2.0 mg/kg,about 4.0 mg/kg, about 5.0 mg/kg, about 8.0 mg/kg, about 10 mg/kg, about15 mg/kg, about 16 mg/kg, about 20 mg/kg, about 25 mg/kg, or about 30mg/kg body weight. Dose escalation for an individual patient can occurat the discretion of the prescriber in the absence of any clinicallysignificant occurrence that the prescriber might reasonably believewould present an undue safety risk for the patient, such as, forexample, Grade ≧3 non-hematologic toxicity, Grade ≧3 nausea, vomiting ordiarrhea uncontrolled by maximum antiemetic/anti-diarrhea therapy, Grade4 neutropenia lasting >7 days in the absence of growth factor support,Grade 3 or 4 neutropenia of any duration accompanied with fever ≧38.5°C. and/or systemic infection, or other Grade ≧4 hematologic toxicity.

Antibody is usually administered on multiple occasions. An exemplarytreatment regime entails administration once per every two weeks, once amonth, or once every 3 to 6 months. For example, patients can receivethe antibody formulation once every four weeks as a cycle, for exampleevery twenty-eight days. The dosing frequency can be adjusted dependingon the pharmacokinetic profile of the antibody formulation in thepatient. For example, the half-life of the antibody may warrant a twoweek frequency of dosing. In some methods, two or more monoclonalantibodies with different binding specificities are administeredsimultaneously, in which case the dosage of each antibody administeredfalls within the ranges indicated. Intervals between single dosages canbe weekly, monthly or yearly. Intervals can also be irregular asindicated by measuring blood levels of antibody to amyloid protein(e.g., AA) in the patient. In some methods, dosage is adjusted toachieve a plasma antibody concentration of about 1-1000 μg/ml or about25-300 μg/ml. Alternatively, antibody can be administered as a sustainedrelease formulation, in which case less frequent administration isrequired.

Dosage and frequency vary depending on the half-life of the antibody inthe patient. In general, human antibodies show the longest half life,followed by humanized antibodies, chimeric antibodies, and nonhumanantibodies. The dosage and frequency of administration can varydepending on whether the treatment is prophylactic or therapeutic. Inprophylactic applications, a relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somepatients continue to receive treatment for the rest of their lives. Intherapeutic applications, a relatively high dosage at relatively shortintervals is sometimes required until progression of the disease isreduced or terminated, until a partial or complete response is achieved,and/or until the patient shows lessening or amelioration of symptoms ofdisease. Thereafter, the patent can be administered a prophylacticregime.

The formulations disclosed herein may be provided in a dosage form thatis suitable for parenteral (e.g., intravenous, intramuscular,subcutaneous) administration. As appropriate for particularapplications, the formulation may be alternately provided in a dosagesuitable for rectal, transdermal, nasal, vaginal, inhalant, ocular orother administration. The pharmaceutical formulations are typicallyprepared according to conventional pharmaceutical practice. See e.g.,Remington: The Science and Practice of Pharmacy, (19th ed.) ed. A. R.Gennaro, 1995, Mack Publishing Company, Easton, Pa. and Encyclopedia ofPharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan,1988-1999, Marcel Dekker, N.Y.

In one aspect of the invention, a method of therapeutically orprophylactically treating a human patient having or at risk for havinglight chain (AL) amyloidosis characterized by the presence of amyloidfibrils, deposits or prefibrillar aggregates, comprises administering tothe patient an effective dosage of a pharmaceutical formulationcomprising: (a) an antibody comprising a light chain comprising an aminoacid sequence set forth as SEQ ID NO: 13 and a heavy chain comprising anamino acid sequence set forth as any one of SEQ ID NOs: 14-16, and whichis present at a concentration of about 10 mg/mL; (b) a histidine bufferpresent at a concentration of about 25 mM; (c) trehalose present at aconcentration of about 230 mM; (d) polysorbate 20 present at aconcentration of about 0.2 g/L; and (e) a pH of about 6.5. In such amethod, the dosage is typically from about 0.5 mg/kg to about 30 mg/kgof the antibody (e.g., about 0.5 mg/kg to about 8 mg/kg, or about 8mg/kg to about 30 mg/kg) administered intravenously or subcutaneously ata frequency of from about weekly to about quarterly (e.g., once every 28days).

II.C. Combinational Drug Therapy Treatment Regimes

The present invention also encompasses combination therapies fortreatment or prophylaxis of amyloid disease, particularly AA amyloidosisand AL amyloidosis. Such combination therapies are performed byadministering an antibody formulation of the invention in conjunctionwith one or more second therapeutic agents, such as another therapy totreat or effect prophylaxis of AA amyloidosis or AL amyloidosis, as thecase may be. Combination therapy according to the invention may also beperformed in conjunction with a second therapy is used to treat oreffect prophylaxis of a disease or condition associated with amyloiddisease, such as an inflammatory disease, a chronic microbial infection,a neoplasm (including malignant neoplasms), an inherited inflammatorydisease, and/or a lymphoproliferative disorder. Numerous treatments areavailable in commercial use, in clinical evaluation, and in pre-clinicaldevelopment, any of which could be selected for use in combination withthe disclosed antibody formulations. Such treatments can be one or morecompounds or treatments selected from, but not limited to several majorcategories, namely, (i) non-steroidal anti-inflammatory drugs (NSAIDs;e.g., detoprofen, diclofenac, diflunisal, etodolac, fenoprofen,flurbiprofen, ibuprofen, indomethacin, ketoprofen, meclofenameate,mefenamic acid, meloxicam, nabumeone, naproxen sodium, oxaprozin,piroxicam, sulindac, tolmetin, celecoxib, rofecoxib, aspirin, cholinesalicylate, salsalte, and sodium and magnesium salicylate); (ii)steroids (e.g., cortisone, dexamethasone, hydrocortisone,methylprednisolone, prednisolone, prednisone, triamcinolone); (iii)DMARDs, i.e., disease modifying antirheumatic drugs (e.g., cyclosporine,azathioprine, methotrexate, leflunomide, cyclophosphamide,hydroxychloroquine, sulfasalazine, D-penicillamine, minocycline, andgold); (iv) recombinant proteins (e.g., ENBREL® (etanercept, a solubleTNF receptor) and REMICADE® (infliximab) a chimeric monoclonal anti-TNFantibody); (v) stem cell transplantation; and/or (vi) chemotherapy.Patients with AL amyloidosis may also receive treatment regimes thatinclude drugs or combinations of drugs often used to treat hematologicalmalignancies, such as melphalan, prednisone, dexamethasone, lenalidomide(REVLIMID®) and proteosome inhibitors such as bortezomib (VELCADE®), andcarfilzomib (KYPROLIS™), at dosages in the range of the standard ofcare.

The duration of the combination therapy depends on the type of amyloiddisease being treated, any underlying disease associated with theamyloid disease, the age and condition of the patient, the stage andtype of the patient's disease, how the patient responds to thetreatment, etc. A medical doctor can observe the therapy's effectsclosely and make any adjustments as needed. Additionally, a personhaving a greater risk of developing AA amyloidosis (e.g., a person whois genetically predisposed or previously had an inflammatory disorder orother underlying diseases) or AL amyloidosis may receive prophylacticcombination treatments to inhibit or delay the development of AA ALaggregates such as fibrils, or as maintenance therapy post-treatment.

When performing a combination therapy, the two or more drug substancesare administered simultaneously or sequentially in any order, i.e., aformulation of the invention is administered prior to administering asecond drug substance, concurrently with a second drug substance, orsubsequent to administration of a second drug substance. For example, acombination therapy may be performed by administering a first therapyprior to (e.g., 1 minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes,1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g.,1 minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or12 weeks after) administering a second agent/therapy.

The dosage, frequency and mode of administration of each component ofthe combination can be controlled independently. For example, onetherapeutic agent/therapy may be administered orally three times perday, while the second therapeutic agent/therapy may be administeredintramuscularly once per day. Combination therapy may be given inon-and-off cycles that include rest periods. The compounds may also beadmixed or otherwise formulated together such that one administrationdelivers both compounds. In this case, each therapeutic agent isgenerally present in an amount of 1-95% by weight of the total weight ofthe composition. Alternatively, an antibody formulation of the inventionand a second therapeutic agent can be formulated separately and inindividual dosage amounts. Drug combinations for treatment can beprovided as components of a pharmaceutical pack.

Preferably, the disclosed combination therapies elicit a synergistictherapeutic effect, i.e., an effect greater than the sum of theirindividual effects or therapeutic outcomes. Measurable therapeuticoutcomes are described herein. For example, a synergistic therapeuticeffect may be an effect of at least about two-fold greater than sum ofthe therapeutic effects elicited by the single agents of a givencombination, or at least about five-fold greater, or at least aboutten-fold greater, or at least about twenty-fold greater, or at leastabout fifty-fold greater, or at least about one hundred-fold greater. Asynergistic therapeutic effect may also be observed as an increase intherapeutic effect of at least 10% compared to the sum of thetherapeutic effects elicited by the single agents of a givencombination, or at least 20%, or at least 30%, or at least 40%, or atleast 50%, or at least 60%, or at least 70%, or at least 80%, or atleast 90%, or at least 100%, or more. A synergistic effect is also aneffect that permits reduced dosing of therapeutic agents when they areused in combination.

EXAMPLES

The following examples have been included to illustrate modes of theinvention. Certain aspects of the following examples are described interms of techniques and procedures found or contemplated by the presentco-inventors to work well in the practice of the invention. In light ofthe present disclosure and the general level of skill in the art, thoseof skill appreciate that the following examples are intended to beexemplary only and that numerous changes, modifications, and alterationsmay be employed without departing from the scope of the invention.

Example 1 Selection of Humanized 2A4 for the Treatment of AL Amyloidosis

An IgG1, kappa isotype antibody was prepared, which is a humanizedversion of murine antibody 2A4. The light chain and heavy chainsequences of representative humanized 2A4 antibodies are set forth inFIGS. 1A-1B and 3. Nucleic acids encoding the particular humanized 2A4antibody version 3, which amino acid sequences are shown in FIG. 3, aredepicted in FIGS. 4A-4B.

The parent monoclonal 2A4 antibody is directed against a neo-carboxyterminal epitope of human serum Amyloid A (sAA), resulting from cleavageof the native sAA molecule at amino acid residue 76. The murine antibodydoes not cross-react with IgGs or free light chain (LC) and it has shownbroad isotype recognition of patient derived AL amyloid samples examinedto date. 2A4 recognizes multiple forms of AL light chain amyloidincluding soluble multimer and insoluble deposits. In addition, theantibody has been shown to promote regression of amyloidoma in a mousexenograft model. The light chain and heavy chain sequences of murine 2A4antibody are set forth in FIG. 2.

Example 2 Dose Determination for Humanized 2A4 Antibody

Nonclinical studies in the TRIAD mouse model and the cynomolgus monkeyhave utilized doses of 4 and 40 mg/kg in the mouse and 10, 50, and 100mg/kg in the monkey. Conversion to the Human Equivalent Dose (HED) on amg/kg basis (most appropriate conversion for monoclonal antibodies dueto their restriction to the vascular space) gives HEDs of 0.32 and 3.2for the mouse and 3.2, 16, and 32 for the monkey. Based on currentlyavailable data, the NOAEL in both species is expected to be the highestdose administered. Using a mouse HED (most sensitive species due todosing limitations) of 3.2 and a 10× safety factor, the MRSD for firstin man dosing would be approximately 0.32 mg/kg. Based upon animalstudies, administration to humans is begun with a dose of 0.5 mg/kg.

Example 3 Preparation of the Expression Vector

For generation of the final h2A4 IgG1 HC vector the variable region ofthe heavy chain was isolated by PCR using the plasmidCET1019AS-hygro-h2A4VH3-Sce 4.23.07 as template. Primers used for theamplification introduced at the 5′ end of the fragments an MfeIrestriction site and at the 3′ end a BlpI restriction site forsubcloning. The variable region was cloned into the MfeI and BamHIdigested eukaryotic expression vector pBI-61, which contains the genomicconstant regions of human IgG1 of G1m(3) allotype. The resultingrecombinant expression vector pBI-61/2A4 IgG 1-REM is 9,015 base pairsin size and carries the selectable marker dihydrofolate reductase (DHFR)from hamster under the control of the DHFR promoter and polyadenylationsignal. This vector also contains the beta-lactamase gene for selectionin E. coli as well as origins of replication for E. coli (ColE1 ori),SV40 (SV40 ori) and filamentous phage f1 (f1 ori). Expression of the HCis driven by the immediate early promoter/enhancer region from humancytomegalovirus (CMV) combined with a transcription enhancing element(TE) derived from the hamster genome. For transcript termination andstabilization the polyadenylation signal from hamster growth hormone isused and for enhancement of transcription a non-coding sequence derivedfrom the hamster genome (TE).

Using the plasmid CET1019AS-hu2A4VL3-hck-euro-Sce 4.19.07 as templatethe variable region of the h2A4 LC was isolated by PCR introducing atthe 5′ end of the fragments an SgrAI restriction site and at the 3′ enda KpnI restriction site for subcloning into the final eukaryoticexpression vector pBI-60 digested with the same restriction enzymes.This vector contains the genomic constant region of a human kappa chain.The resulting recombinant expression vector pBI-60/2A4 LC is 7,144 basepairs in size and contains the selectable marker neomycinphosphotransferase mutant, which confers resistance to geneticin, underthe control of the SV40 promoter. For transcript termination thepolyadenylation signal from Herpes simplex thymidine kinase is used.This vector also contains the beta-lactamase gene for selection in E.coli as well as origins of replication for E. coli (ColE1 ori) andfilamentous phage f1 (f1 ori). Expression of the LC is driven by theimmediate early promoter/enhancer region from human cytomegalovirus(CMV) combined with a transcription enhancing element (TE) derived fromthe hamster genome. For transcript termination and stabilization thepolyadenylation signal from hamster growth hormone is used and forenhancement of transcription a non-coding sequence derived from thehamster genome (TE).

Example 4 Production of Humanized 2A4 Antibody (Pool-Derived Material)

Humanized 2A4 was produced in Chinese Hamster Ovary (CHO) cells, grownin chemically defined media without any bovine-derived components.Antibody was pooled from stable transfected cells from which theproduction cell line was ultimately derived. The pool-derived materialwas purified by protein A-affinity chromatography. This material wasused for human tissue cross-reactivity studies and for a single dosepharmacokinetic (PK) study in cynomolgus monkeys. The formulation of thehumanized 2A4 antibody is 10 mg/mL antibody, 25 mML-Histidine/L-Histidine HCl monohydrate, 230 mM Trehalose dehydrate,0.02% (w/v) Polysorbate (TWEEN®) 20, pH=6.5.

Example 5 Production of Humanized 2A4 Antibody (Clone-Derived Material)

A single CHO cell clone was isolated from cell pools as described inExample 3, and was used to establish the Master Cell Bank (MCB) withoutany bovine materials. Humanized 2A4 for nonclinical studies wasmanufactured at 80 L scale using the same cell cultivation andpurification processes (except scale-up modifications) as the GMPclinical version of humanized 2A4 (2,000 L scale). Material from the2,000 L scale production may also be used in nonclinical studies.

Example 6 Process of Manufacturing Humanized 2A4 Antibody

Vial Thaw & Inoculum Expansion.

Cells from the MCB are thawed and transferred into an appropriate cellculture flask. The cells are incubated at approximately 37° C. Thethawed culture is propagated for one to four days (first passage aftercell thaw). For sub-cultivation, an aliquot of a grown cell culture (anda defined volume of pre-warmed, 0.22 μm or less filtered inoculummedium) is used to reach a seed density of approximately 0.1-0.5×10⁶cells/mL in standard cell culture vessels of approximately 0.02 L to 1 Lworking volume. As an example, the first passages can be done in 0.125 Lor 0.25 L or 0.5 L vessels, followed by passages in 1 L vessels. A stockculture can be initiated at this cultivation stage. For preparation ofinoculum cultures for individual production fermenters, aliquots of thestock cultures are expanded to generate cultures with up to 25 L volume.Typically, the cell culture is scaled up from 1 L cultures to 2 or more1 L or 2 L cultures, then to 2 or more 2 L or 3 L cultures and finallyto 2 or more cultures with up to 25 L culture volume per vessel. Growncell suspensions from several vessels can be pooled and used toinoculate the 80 L bioreactor. Shake flasks, T-flasks, spinner flasksand bags can be used as standard cell culture vessels for the abovecultivation steps.

Seed Cultures in Bioreactors.

Before inoculation with cells, 0.22 μm or less filtered growth medium isadded to the bioreactors. The content of the filled bioreactors iswarmed to approximately 37° C. and maintained at this temperaturethroughout incubation of the cells. Cells from the inoculum cultures aretransferred into the pre-warmed medium. The initial cell density istargeted within the range of 0.1-0.5×10⁶ cells/mL. The cells are grownin an 80 L bioreactor and subsequently in a 400 L bioreactor. Cells aresubcultivated approximately every two to four days. At this stage, cellsmay be transferred to another vessel of the same or larger volume.Typically, the cell culture is scaled up from 1×80 L bioreactor cultureto 1×400 L culture. To initiate the production phase, the cells aretransferred from the grown 400 L cell suspension to the productionbioreactor of approximately 2,000 L working volume.

Production Culture in 2,000 L Bioreactor.

Before inoculation with cells, 0.22 μm or less filtered productionmedium is added to the production bioreactor. The content of the filledproduction bioreactor is warmed to approximately 37° C. and maintainedat this temperature throughout incubation of the cells. The initial celldensity in the production phase is targeted within the range of0.1-0.5×10⁶ cells/mL. The production bioreactor is run in a fed batchmode. To support the production of antibody and to prolong cultureduration, a nutrient feed medium is added during the production stage.The point at which to start feeding is determined either by culture timeor by cell density. As needed, a glucose solution and/or glutaminesolution can be added during the production stage to avoid depletion ofthese substances during the production period. The run time of the 2,000L production bioreactor is typically 8 to 14 days. Pre-harvest samplesare tested for sterility, mycoplasma, and adventitious virus in vitro.

Harvest and Clarification.

After 8 to 14 days of cultivation in the production phase, the cellculture fluid is separated from the cells. After pre-harvest samplingand prior to harvest, the pH and the temperature of the culture can beadjusted to facilitate removal of cells, debris and particles duringharvest. To remove the cells, the culture is passed through acentrifugation plus dead-end filtration unit. The cells are centrifugedand/or retained by the membranes. The harvested culture fluid is passedthrough filters of 0.22 μm pore size or less and collected in anappropriate container. Residual culture fluid can be removed from theharvest system by flushing with Phosphate Buffered Saline (PBS) torecover residual product from the harvest system. The resultingrecovered product amount is collected together with the harvestedculture fluid to form the harvest pool, also called harvested cell-freeculture fluid (HCCF). The pH and temperature of the HCCF can be adjustedto facilitate the subsequent downstream processing steps.

Purification.

The antibody is purified from the HCCF by a series of steps involvingaffinity chromatography, acid treatment, depth filtration, anionexchange chromatography, cation exchange chromatography, nanofiltrationand ultra-/diafiltration, several of which may be performed in severalcycles. To remove contaminants the affinity chromatography process stepspecifically binds the antibody product. The HCCF is applied to thechromatography column packed with the MabSelect matrix. The matrix bindsantibody at neutral pH, while contaminants appear in the flow throughand are removed. The column is eluted in a step elution with a 100 mMacetic acid/sodium acetate solution at pH 3.5. To inactivate potentialviral contaminants, the antibody solution is incubated at roomtemperature for a minimum of 60 minutes at pH 3.5±0.1. After incubationthe acid treated pool is adjusted to pH 7.2 using a 2 M Trometamolsolution and subjected to depth filtration for clarification. For anionexchange chromatography, the depth filtered product pool is adjusted toa conductivity ≦7 mS/cm with Water for Injection (WFI). The adjustedpool is applied to a chromatography column packed with Q Sepharose FFresin. The antibody passes through the anion exchange matrix unbound.The flow through is monitored and the antibody containing fraction iscollected based on absorbance measurement. For cation exchangechromatography, the product pool is adjusted to a pH of 5.5±0.1 byaddition of acetic acid up to a conductivity of ≦7.5 mS/cm with WFI. Theadjusted product pool is applied onto a chromatography column packedwith SP Sepharose FF cation exchange resin. This chromatography step isperformed in a bind-elute mode. The antibody binds to the cationexchange matrix. The column is eluted in a step elution with a 100 mMacetic acid/sodium acetate and 138.5 mM sodium chloride solution at pH5.5. Potential viral contaminants are removed by passing the antibodysolution through a 0.1 μm prefilter and a Planova 20N nanofilter at amaximum pressure of 1 bar differential pressure of the Planova 20Nnanofilter. During ultrafiltration/diafiltration (UF/DF), the product isconcentrated to the target concentration, and the buffer is exchangedwith the formulation buffer. Concentration and diafiltration isperformed using ultrafiltration membranes having a cut-off ofapproximately 30 kD. The material is processed by concentrating theproduct to 30-100 mg/mL. The 30 kD pool is then diafiltered with asolution of 25 mM L-Histidine, pH 6.5 and is flushed to a concentrationof about 60-70 mg/mL. The 30 kD pool intermediate may be stored at −40°C. until formulation is performed. For formulation, the 30 kD productpool is adjusted to a solution containing 17.5 mM L-Histidine/7.5 mML-Histidine Hydrochloride, 230 mM Trehalose, and 0.02% (w/v)Polysorbate20, pH=6.5. The antibody is finally diluted with formulationbuffer to the desired target concentration of 10 mg/mL. The resultingdrug substance is filtered through a 0.22 μm filter to remove anypotential adventitious microbial contaminants and particulate material.The drug substance can be stored frozen at −40° C. until filling.

Example 7 Characterization of Drug Substance Containing Humanized 2A4Antibody

Humanized 2A4 used for formulation is composed of two heterodimers. Eachof the heterodimers is composed of a heavy polypeptide chain of ˜50 kDa(449 amino acids) and a kappa light polypeptide chain of ˜24 kDa (219amino acids). The antibody protein has a humanized amino acid sequencewith a total molecular mass of approximately 147 kDa. The fourpolypeptide chains of the antibody molecule are linked together bydisulfide bonds. Each heavy polypeptide chain contains one consensussequence for N-linked glycosylation, which is occupied (positions 299 to301, highlighted in bold and underlining in FIG. 1A). There are twobinding sites for the serum amyloid A epitope per antibody molecule.

A competitive binding ELISA has been established to measure binding ofhumanized 2A4 to its antigen (CGGHEDT (SEQ ID NO: 17) when conjugated toOvalbumin) compared to the reference standard.

Example 8 Humanized 2A4 Drug Substance Components and Composition

The humanized 2A4 drug substance (100 mg/vial) for clinical use is asterile liquid dosage form consisting of a 10 mL fill in a 25 mL vial(20R). The nonclinical humanized 2A4 drug substance (200 mg/vial) is 20mL fill in a 25 mL vial (20R). The nonclinical and clinical formulationsof humanized 2A4 are provided in Table 1. The final formulation of thehumanized 2A4 drug substance has a density of 1.034 g/mL at 20° C. and apH of 6.5.

TABLE 1 Composition of Nonclinical and Clinical Humanized 2A4 DrugSubstance Nominal amount (mg/vial) Concen- Nonclinical Clinical trationVial Size = Vial Size = Component Function (g/L) 25 mL (20R) 25 mL (20R)Humanized 2A4 Active 10 200 100 drug substance Substance L-HistidineBuffer 2.72 54.4 27.2 component L-Histidine HCl Buffer 1.57 31.4 15.7monohydrate component Trehalose Tonicity 87.02 1,740.4 870.2 dihydrateagent Polysorbate Surfactant 0.20 4.0 2.0 (TWEEN ®) 20 Water for Solvent— Add WFI Add WFI Injection (WFI) to a total to a total volume of volumeof 20 mL 10 mL

Example 9 Batch Formula for Drug Product (100 Mg/Ml Vial)

A formula was designed for a 2,600 vial batch of drug product asprovided in Table 2.

TABLE 2 Batch Formula for 2,600 Vials Ingredient Grade Quantity perBatch Humanized 2A4 antibody — 260.0 g L-Histidine USP, Ph. Eur. 70.72 gL-Histidine HCl monohydrate Ph. Eur. 40.82 g Trehalose dehydrate USP/NF,Ph. Eur. 2,262.52 g   Polysorbate 20 USP/NF, Ph. Eur.  5.20 g

Example 10 Lyophilization

A Hof Com 26041 freeze dryer was used to lyophilize the formulatedhumanized 2A4 drug substance over a period of approximately 86 hourswith the pressure regulated by an MKS control system (MKS Instruments)with N₂ injection according to the program set forth in Table 3. Theendpoint was detected by Pirani signal. During the drying mode, thevials stand directly on the shelves without lyo plates. The nitrogenbackfill is at approximately 600 mbar with pharma grade, sterile N₂. Thevials were then closed and stored at 5° C. within the freeze dryer. Thefinal drug product is stored at 2-8° C., protected from light. Theprocess should yield a white to yellowish lyo cake.

Table 3 summarizes the program for the lyophilization of humanized 2A4drug substance.

TABLE 3 Lyophilization Steps Time Shelf temperature Vacuum MKS Step StepNo. [hh:mm] [° C.] [mbar] Loading 01 00:01 5 off Freezing 02 00:15 5 off03 00:05 2 off 04 02:00 2 off 05 01:05 −50 off 06 02:30 −50 off Primary07 00:05 −50 0.10 Drying 08 00:40 −10 0.10 09 55:00 −10 0.10 Secondary10 04:30 30 0.10 Drying 11 20:00 30 0.10 Total Time 86:11

Example 11 Reconstitution of Lyophilized Drug Product

Prior to application, the lyophlisate has to be reconstituted withsterilized water for injection. The reconstitution of h2A4 vials hasbeen performed according to the following procedure under laminarair-flow. The complete flip-off-cap of the respective product vial wasremoved. The rubber-stopper was also removed. The solvent was added bypipetting the necessary volume (2×5 mL WFI using a piston pipette). Whenperforming this action, it was ensured that the solvent was added slowlyto the lyophilized product. The vials were carefully swirled (notshaken), until the lyophilized product was completely dissolved. Thesolution was made homogenous by carefully rotating the vialend-over-end. The dissolved material was aliquoted according to table 1and stored at −70° C. until analysis

Example 12 Clinical Assessment of Humanized 2A4 Drug Substance

A clinical trial is designed to determine a maximum tolerated dose (MTD)and/or the Phase 2 recommended dose (P2RD) of humanized 2A4 drugsubstance in subjects with AL amyloidosis. Dosing will begin at 0.5mg/kg and escalate to a high of 30 mg/kg or 2500 mg total (whichever islower). Initially, humanized 2A4 drug substance will be givenintravenously as a single agent every 28 days until progression of organfunction or unacceptable treatment related toxicity or withdraw ofconsent. If the half-life (t_(1/2)) of humanized 2A4 drug substance fromthe initial doses suggests that a different dosing schedule would bemore appropriate (e.g., every two weeks or an alternate, less frequentschedule than once every 28 days), dosing in subsequent cohorts may bemodified using an alternative dosing schedule.

1. A pharmaceutical formulation comprising: (a) a chimeric or humanizedversion of antibody 2A4 (ATCC Accession Number PTA-9662) or of antibody7D8 (ATCC Accession Number PTA-9468), or fragment thereof, thatspecifically competes for binding to antigen with 2A4 or 7D8, whereinthe antibody is present at a concentration within the range from about 1mg/mL to about 100 mg/mL; (b) histidine buffer present at aconcentration within the range from about 20 mM to about 30 mM; (c)trehalose present at a concentration within the range from about 210 mMto about 250 mM; and (d) polysorbate 20 present at a concentrationwithin the range from about 0.005% to about 0.05% by weight; wherein theformulation is characterized by a pH within the range from about 6 toabout
 7. 2. The formulation of claim 1, wherein the antibody is ahumanized version of antibody 2A4.
 3. The formulation of claim 1,wherein the antibody comprises a light chain variable region comprisingan amino acid sequence set forth as SEQ ID NO:
 4. 4. The formulation ofclaim 1, wherein the antibody comprises a heavy chain variable regioncomprising an amino acid sequence set forth as SEQ ID NO:
 5. 5. Theformulation of claim 3, wherein the antibody comprises a heavy chainvariable region comprising an amino acid sequence set forth as SEQ IDNO:
 5. 6. The formulation of claim 5, wherein the antibody comprises alight chain comprising an amino acid sequence set forth as SEQ ID NO: 13and a heavy chain comprising an amino acid sequence set forth as any oneof SEQ ID NO: 14-16.
 7. The formulation of claim 6, wherein the antibodycomprises a light chain comprising an amino acid sequence set forth asSEQ ID NO: 13 and a heavy chain comprising an amino acid sequence setforth as SEQ ID NO:
 15. 8. The formulation of claim 1, wherein theantibody comprises a light chain variable region comprising threecomplementarity determining regions set forth as SEQ ID NOs: 6, 7, and8, and a heavy chain variable region comprising three complementarityregions set forth as SEQ ID NOs: 9, 10, and
 11. 9. The formulation ofclaim 1, wherein the antibody is a humanized or chimeric version ofantibody 7D8 produced by ATCC Accession Number PTA-9468.
 10. Theformulation of claim 1, wherein the antibody comprises a light chainvariable region comprising three complementarity determining regions setforth as SEQ ID NOs: 12, 7, and 8, and a heavy chain variable regioncomprising three complementarity regions set forth as SEQ ID NOs: 9, 10,and
 11. 11. The formulation of any one of claims 1, wherein the antibodyis present at a concentration within the range from about 5 mg/mL toabout 15 mg/mL.
 12. The formulation of claim 11, wherein the antibody ispresent at a concentration of about 10 mg/mL.
 13. The formulation of anyone of claims 1, wherein the antibody is present at a concentrationwithin the range from about 25-75 mg/mL.
 14. The formulation of claim13, wherein the antibody is present at a concentration of about 50mg/mL.
 15. The formulation of any one of claims 1, wherein the histidinebuffer is present at a concentration of about 25 mM.
 16. The formulationof claim 15, wherein the histidine buffer comprises L-histidine andL-histidine HCl monohydrate.
 17. The formulation of claim 16, whereinthe L-histidine is present at a concentration within the range fromabout 16 mM to about 22 mM and the L-histidine HCl monohydrate ispresent at a concentration within the range from about 4 mM to about 8mM.
 18. The formulation of any one of claims 1, wherein the trehalose ispresent at a concentration of about 230 mM.
 19. The formulation of anyone of claims 1, which is characterized by an osmolality of about 300mOsm/kg.
 20. The formulation of any one of claims 1, wherein less thanabout 10% of the antibody is present as an aggregate in the formulation.21. The formulation of any one of claims 1, which further comprises abulking agent.
 22. The formulation of any one of claims 1, which issterile.
 23. The formulation of any one of claims 1, which is stableupon freezing and thawing.
 24. A pharmaceutical formulation comprising:(a) an antibody comprising a light chain comprising an amino acidsequence set forth as SEQ ID NO: 13 and a heavy chain comprising anamino acid sequence set forth as any one of SEQ ID NOs: 14-16, and whichis present at a concentration of about 10 mg/mL; (b) a histidine bufferpresent at a concentration of about 25 mM; (c) trehalose present at aconcentration of about 230 mM; (d) polysorbate 20 present at aconcentration of about 0.2 g/L; and (e) a pH of about 6.5.
 25. Theformulation of claim 24, wherein the antibody comprises a light chaincomprising an amino acid sequence set forth as SEQ ID NO: 13 and a heavychain comprising an amino acid sequence set forth as SEQ ID NO:
 15. 26.A pharmaceutical formulation comprising: (a) an antibody, which isantibody 2A4 (ATCC Accession Number PTA-9662), antibody 7D8 (ATCCAccession Number PTA-9468), or a chimeric or humanized version ofantibody 2A4 or of antibody 7D8, or fragment thereof, that specificallycompetes for binding to antigen with 2A4 or 7D8, wherein the antibody ispresent at a concentration within the range from about 50 mg/mL to about100 mg/mL; (b) a buffer; (c) a non-reducing sugar; and (d) a non-ionicsurfactant.
 27. A lyophilized formulation of an antibody, comprising (a)a humanized version of antibody 2A4 (ATCC Accession Number PTA-9662) orantibody 7D8 (ATCC Accession Number PTA-9468) or antigen bindingfragment thereof; (b) histidine; (c) trehalose; and (d) polysorbate 20.28. The lyophilized formulation of claim 27, wherein the formulation hasa pH of between about 6 to about 7 when reconstituted.
 29. Thelyophilized formulation of claim 28, wherein the formulation has a pH ofabout 6.5 when reconstituted.
 30. The lyophilized formulation of claim27, comprising about 100 mg to about 1000 mg of the antibody.
 31. Thelyophilized formulation of claim 27, wherein the polysorbate 20 ispresent at a concentration within the range from about 0.005% to about0.05% by weight.
 32. The lyophilized formulation of claim 27, thatenables reconstitution to yield an aqueous solution comprising: (a) anantibody comprising a light chain comprising an amino acid sequence setforth as SEQ ID NO: 13 and a heavy chain comprising an amino acidsequence set forth as any one of SEQ ID NOs: 14-16, and which is presentat a concentration of about 10 mg/mL; (b) a histidine buffer present ata concentration of about 25 mM; (c) trehalose present at a concentrationof about 230 mM; (d) polysorbate 20 present at a concentration of about0.2 g/L; and (e) a pH of about 6.5.
 33. The lyophilized formulation ofclaim 32, wherein the lyophilized formulation comprises about 100 mg ofthe antibody and enables reconstitution with sterile water. 34-36.(canceled)
 37. A nucleic acid comprising a nucleotide sequence encodingany one of SEQ ID NOs: 13, 14-16, 19, 20, 22 or
 23. 38-40. (canceled)41. A vector comprising at least one nucleic acid of claim
 37. 42-44.(canceled)
 45. A host cell having stably incorporated into its genome atleast one nucleic acid of claim
 37. 46. The host cell of claim 45,wherein the host cell is a CHO cell.
 47. (canceled)
 48. The host cell ofclaim 38 having stably incorporated into its genome a nucleic acidcomprising the nucleotide sequence encoding SEQ ID NO: 19 and a nucleicacid comprising the nucleotide sequence encoding SEQ ID NO:
 22. 49. Thehost cell of claim 38 having stably incorporated into its genome anucleic acid comprising the nucleotide sequence encoding SEQ ID NO: 20and a nucleic acid comprising the nucleotide sequence encoding SEQ IDNO:
 23. 50. (canceled)
 51. A method of making a pharmaceuticalformulation comprising: (a) culturing mammalian cells having stablyincorporated into their genome one or more nucleic acids encoding thelight and heavy chains of a humanized 2A4 antibody so that the cellssecrete the antibody into the cell culture media, and purifying theantibody from the cell culture media; (b) and preparing the formulationof claim
 1. 52. (canceled)
 53. The method of claim 52, wherein thenucleic acid encoding the light chain of a humanized 2A4 antibodycomprises a nucleotide sequence encoding SEQ ID NO: 13, and wherein thenucleic acid encoding the heavy chain of a humanized 2A4 antibodycomprises a nucleotide sequence encoding SEQ ID NO:
 15. 54. The methodof claim 53, wherein the nucleic acid encoding the light chain of ahumanized 2A4 antibody comprises the nucleotide sequence of SEQ ID NO:19, and wherein the nucleic acid encoding the heavy chain of a humanized2A4 antibody comprises the nucleotide sequence of SEQ ID NO:
 22. 55. Themethod of claim 54, wherein the nucleic acid encoding the light chain ofa humanized 2A4 antibody comprises the nucleotide sequence of SEQ ID NO:20, and wherein the nucleic acid encoding the heavy chain of a humanized2A4 antibody comprises the nucleotide sequence of SEQ ID NO:
 23. 56-57.(canceled)
 58. A method of therapeutically or prophylactically treatinga human patient having or at risk for having amyloidosis characterizedby the presence of amyloid protein fibrils, the method comprisingadministering to the patient an effective dosage of the formulation ofclaim
 1. 59. The method of claim 58, wherein the human patient hasamyloid A amyloidosis characterized by the presence of amyloid A proteinfibrils.
 60. The method of claim 59, wherein the formulation is theformulation of claim
 25. 61. The method of claim 60, wherein the humanpatient has AL amyloidosis characterized by the presence of amyloidlight chain-type protein fibrils.
 62. (canceled)
 63. The method of claim61, wherein the patient is also treated with one or both of melphalan,bortezomib, lenolidomide or carfilzomib.
 64. The method of claim 63,wherein the patient is treated with melphalan and/or bortezomib prior totreatment with the formulation.
 65. The method of claim 64, wherein thepatient is treated with melphalan and/or bortezomib concurrently withtreatment with the formulation.
 66. The method of claim 64, wherein thepatient is treated with melphalan and/or bortezomib subsequent totreatment with the formulation.
 67. The method of claim 61, wherein theAL amyloidosis is associated with a dyscrasia of the B lymphocytelineage.
 68. The method of claim 67, wherein the dyscrasia is amalignancy.
 69. The method of claim 68, wherein the malignancy ismultiple myeloma.
 70. The method of claim 61, wherein the formulation isadministered in multiple dosages.
 71. The method of claim 70, whereinthe formulation is administered at a frequency in a range of about dailyto about annually.
 72. The method of claim 71, wherein the frequency isin a range of about every other week to about every three months. 73.The method of claim 61, wherein the formulation is administeredintravenously at a dose in a range from about 10 mg to about 5000 mghumanized 2A4 drug substance.
 74. The method of claim 73, wherein theformulation is administered at a dose in a range from about 30 mg toabout 2500 mg humanized 2A4 drug substance at a frequency in a range ofabout every other week to about every other month.
 75. The method ofclaim 73, wherein the formulation is administered once a month.
 76. Themethod of claim 73, wherein the dose is about 30 mg humanized 2A4 drugsubstance.
 77. The method of claim 73, wherein the dose is about 100 mghumanized 2A4 drug substance.
 78. The method of claim 73, wherein thedose is about 300 mg humanized 2A4 drug substance.
 79. The method ofclaim 73, wherein the dose is about 1000 mg humanized 2A4 drugsubstance.
 80. The method of claim 73, wherein the dose is about 2500 mghumanized 2A4 drug substance.
 81. A method of therapeutically orprophylactically treating a human patient having or at risk for havinglight chain (AL) amyloidosis characterized by the presence of amyloidfibrils, deposits or prefibrillar aggregates, comprising administeringto the patient an effective dosage of a pharmaceutical formulationcomprising: (a) an antibody comprising a light chain comprising an aminoacid sequence set forth as SEQ ID NO: 13 and a heavy chain comprising anamino acid sequence set forth as any one of SEQ ID NOs: 14-16, and whichis present at a concentration of about 10 mg/mL; (b) a histidine bufferpresent at a concentration of about 25 mM; (c) trehalose present at aconcentration of about 230 mM; (d) polysorbate 20 present at aconcentration of about 0.2 g/L; and (e) a pH of about 6.5.
 82. Themethod of claim 81, wherein the dosage is from about 0.5 mg/kg to about30 mg/kg of the antibody administered intravenously or subcutaneously ata frequency of from about weekly to about quarterly.
 83. The method ofclaim 82, wherein the frequency of administration is once every 28 days.84. The method of claim 82, wherein the dosage is about 0.5 mg/kg toabout 8 mg/kg.
 85. The method of claim 82, wherein the dosage is about 8mg/kg to about 30 mg/kg.
 86. A pharmaceutical product, comprising: (a) avial comprising about 100 mg antibody in powder form; (b) instructionsfor reconstitution of the antibody; and (c) instructions for preparingthe reconstituted antibody for infusion, wherein: (i) the antibodycomprises a light chain comprising an amino acid sequence set forth asSEQ ID NO: 13 and a heavy chain comprising an amino acid sequence setforth as any one of SEQ ID NOs: 14-16; and (ii) the reconstitutioninstructions require reconstitution with water for injection to anextractable volume of 10 mL.